The thiosemicarbazone of 5-hydroxy-2-formyl pyridine shows excellent antineoplastic activity in animals and recently has undergone clinical tests in humans. It has been hyypothesized that this compound acts as a tridentate ligand to bind iron at the active site of ribonucleoside diphosphate reductase, a key enzyme in the synthesis of DNA precursors, to inhibit catalysis. Further, its toxicity has been attributed in part to a disturbance of iron metabolism. We will examine these proposals (1) with studies t determine the profile of the transition metal chemistry of this and related ligands, (2) with studies of the nature of inhibition of ribonucleotide diphosphate reductase by this compound, (3) with in vitro studies under controlled metal ion conditions to determine the active cytotoxic species, and (4) with investigations of other possible sites of cellular inhibition such as oxidative phosphorylation. Finally, using these appraches we will examine the basis for the variation in biological activity of this compound with substituent changes in the 5-position of the pyridine ring.